Since the first discovery of polymer nanocomposites by Toyota Central R&D Labs., Inc., the researches on polymer nanocomposites have extensively been conducted for various applications. In order to achieve the high functionalities and high performances, nanofillers with high aspect ratio have been expected. Among them, graphene, which is known as a single layer of graphite, have attracted a great deal of attention because of the excellent properties such as mechanical properties, thermal properties, electrical properties and so on. In this study, graphene-based polyamide 66 (PA66) nanocomposites were prepared by in-situ polymerization. Graphene oxide (GO) and chemically modified GO (GO-DDA) were used as fillers to investigate the effect of chemical structure of the fillers on the structure and the properties of the nanocomposites. In the process of the sample preparation, PA66 was polymerized under the presence of the fillers which highly dispersed in the aqueous solvent. The nanocomposites with the thickness of 200 μm were obtained by the melt press of the polymerized products. The structural studies revealed that the fillers were homogeneously dispersed in the nanocomposites at nano-scale. Due to the melt-press, GO and GO-DDA were highly aligned to the in-plane direction. From the FTIR spectra, it was revealed that amide bonds were formed at the interface between PA66 and the fillers. Therefore, the high dispersion of the fillers and the strong interfacial interactions were achieved in the nanocomposites. As a result, the high mechanical properties and the high thermal resistance of the nanocomposites were revealed. It is well known that the rigid fillers have often reduced the elongation at break and the toughness of the composites, resulting in very brittle materials. However, in the present nanocomposites, elongation at break of PA66 was preserved so that the nanocomposites showed favorable toughness. It was assumed that the highly aligned 2D fillers could effectively reduce the stress concentration points and could prevent the crack propagation in the nanocomposites. Furthermore, the nanocomposites showed the strong increase in the Young’s modulus and the tensile strength. The experimental Young’s modulus of the nanocomposites exceeded the theoretical values calculated by Halpin-Tsai model. The Young’s modulus increased by 92% and 42% for the nanocomposites by the addition of only 0.3 wt% of GO-DDA and GO, respectively. As for the thermal resistance, the highest thermal decomposition temperature of 388 °C was achieved by the nanocomposites with GO-DDA 0.5 wt%, that was 31 °C higher than that of PA66. These strong enhancements in the properties indicate that not only the chemical interfacial interactions through covalent bonding but also the physical interactions of molecular chains have the important effects on the reinforcement of the nanocomposites.